Numerous investigators in the selenium field have proposed that low molecular weight selenocompounds are responsible for the numerous health benefits attributed to selenium, while an alternative proposal is that selenoproteins are likely the more responsible agents. These health benefits include preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process and having roles in mammalian development, male reproduction and immune function. We were amongst the first to propose several years ago that these health benefits are due largely to the presence of selenium in selenoproteins as the amino acid, selenocysteine (Sec).Therefore, to elucidate the role of selenoproteins in cancer prevention, we are characterizing the function of different selenoproteins in the malignancy process. There are three selenoproteins, thioredoxin peroxidase 1 (TR1), selenoprotein 15 (Sep15) and glutathione peroxidase 2 (GPx2), that have been shown by us and others to have roles in both preventing and promoting cancer. Our laboratory has taken the lead in investigating two of these selenoproteins, TR1 and Sep15, as discussed below. In our examination of TR1 in promoting cancer, we have used multiple approaches to elucidate the role of this selenoprotein in cancer promotion. Selenite is known to be toxic to mammalian cells and cancer cells are far more sensitive to this toxicity. We previously targeted the removal of both TR1 and thioredoxin 1 (Trx1) in DT cells which are an oncogenic cell line derived from the mouse embryonic fibroblast NIH 3T3 cells and found that TR1-deficient cells were significantly more sensitive to selenite exposure than Trx1-deficient cells or control cells. In the past year, we have completed and published the selenite toxicity project by demonstrating that only TR1-deficient cells manifested strongly enhanced production and secretion of glutathione, which was associated with increased sensitivity of the cells to selenite. The data uncover a new role of TR1 in cancer that is independent of Trx reduction and compensated for by the glutathione system. The data also suggest that the enhanced selenite toxicity of cancer cells and simultaneous inhibition of TR1 can provide a new avenue for cancer therapy.As noted in last year?s report, TR1 and Sep15 have roles in protecting normal cells from cancer and, we have had a major hand in demonstrating that these two selenoproteins also have roles in promoting cancer. We previously published that the targeted removal of Sep15 expression in CT-26 cells, a mouse colon carcinoma cell line, altered several of the malignant characteristics more towards normal cells and have continued these studies to include the targeted knockdown of both TR1 and Sep15 in CT-26 cells. As noted in last year?s report, the down-regulation of either Sep15 or TR1 resulted in reversal of the cancer phenotype in these cells, but surprisingly the double-knockdown of both Sep15 and TR1 appeared to retain the cancer phenotype of the parental cells. In this year, our results indicate that CT-26 cells, lacking either Sep15 or TR1, over-express mRNA levels of cell cycle regulatory genes such as cyclin B1 interacting protein 1, thus causing a delayed entry of the cells into mitosis and, as a result, slowed cell growth. Additionally, microarray analyses indicated a strong increase in guanylate binding proteins and other interferon/inflammation-associated genes in Sep15 knockdown cells. However, this increase was not observed in TR1 knockdown cells or in the cells lacking both Sep15 and TR1. Thus, the mechanism by which cells lacking Sep15 or TR1 reverse the cancer phenotype in cells appears to be very different. We are continuing to investigate the differences between these mechanisms, and furthermore the apparent compensatory effect of simultaneously removing Sep15 and TR1 in colon cancer cells.In addition, the regulation of TR1 expression by hypoxic conditions was also examined in cancer cell lines to further elucidate the role of this selenoprotein under these stressful conditions. Hypoxic conditions have been described in the literature to increase the levels of ROS inside cells and this ROS have been described to be critical for the hypoxic-regulation of HIF stabilization and activity. Due to the importance of some selenoproteins in the regulation of redox balance in the cells, we undertook a project to examine the effect of hypoxic conditions on the expression of selenoproteins. In a previous study, we observed that hypoxia reduced TR1 and its mRNA levels and activity in DT cells and a mouse breast cancer cell line, EMT6. This regulation is independent of the activity of hypoxia inducible factor (HIF) and TR1 over-expression abrogates the increase in ROS levels induced by hypoxia, but does not affect HIF activity or stabilization. In the last year, we completed and published this project. As a continuation of this project, we expanded our finding that Trx1 over-expression increased HIF hypoxic activity in HeLa and EMT6 cell lines, whereas HIF activity or stabilization is not affected in DT, MCF-7 and HT-29 cell lines after Trx1 over-expression. The effect of Trx1 on HIF activity is dependent of the presence of the two cysteine residues that are reduced by thioredoxin reductase activity, as the over-expression of a mutated form of Trx1 did not have any effect on HIF activity. However, the over-expression of TR1 did not recapitulate the effects of Trx1. Furthermore, Trx1 over-expression in EMT6 or in HeLa cells in which TR1 was knocked down was still able to induce and increase in HIF activity under hypoxic conditions, suggesting that other proteins than TR1 are reducing Txn1 in this process. As noted in last year?s report, we reported that TR1 knockdown in EMT6 mouse breast cancer cells caused increased sensitivity to TNF-alpha induced apoptosis. The targeted removal of Trx1 was achieved using siRNA technology, and sensitivity to TNF-alpha induced apoptosis was examined to elucidate the role of the Trx system in apoptosis. Trx1 deficient cells had the highest sensitivity to TNF-alpha and TR1 deficient cells had a higher sensitivity compared to control cells. These observations suggested that the Trx system is involved in an anti-apoptotic pathway. We examined the activation of MAPKs by TNF-alpha treatment to examine the pathway involved in the anti-apoptotic function of the Trx system and found TR1 and Trx1 knockdown cells showed a differential activation of Erk and p-38. In the past year, we found that Erk activation was lower in TR1 and Trx1 knockdown cells after TNF-alpha treatment and inhibition of Erk activation increased caspase 3 activation in all cells (control, TR1 and Trx1). This provided evidence that Erk is involved in the anti-apoptotic pathway in TR1 and Trx1 deficient cells. We also observed that the nuclear localization of p-Erk by TNF-alpha was higher in TR1 and Trx1 knockdown cells and inhibition of PI3K with LY294002 blocked TNF-alpha induced apoptosis in each cell line. Inhibition of PI3K inhibited the nuclear localization of p-Erk by TNF-alpha, whereas it did not inhibit the overall activation of MAPKs suggesting a function of nuclear p-Erk in TNF-alpha induced apoptosis. We are in the process of elucidating the function of nuclear p-Erk in TNF-alpha induced apoptosis.